外泌体来源的miR-1275通过上调淋巴细胞中IL-38的表达抑制脓毒症心肌细胞凋亡

doi:10.12122/j.issn.1673-4254.2025.08.05

摘要/Abstract

摘要：

目的探究心肌细胞来源的外泌体对脂多糖（LPS）诱导的脓毒症心肌细胞损伤的影响及其作用机制。方法采用LPS构建脓毒症心肌细胞损伤模型，分离心肌细胞来源外泌体，与大鼠淋巴细胞共培养。实验分为以下2组：对照组（心肌细胞置于含10%胎牛血清的DMEM培养基中）和LPS组（心肌细胞置于含500 ng/mL LPS的培养基诱导3 h）。利用CCK-8检测淋巴细胞增殖情况；qRT-PCR和Western blotting法检测淋巴细胞中白细胞介素-38（IL-38）的mRNA和蛋白表达水平；通过生物信息学挖掘LPS诱导脓毒症心肌细胞损伤的关键外泌体，双荧光素酶报告验证其与IL-38是否有靶向关系。将LPS诱导的心肌细胞来源外泌体处理后的淋巴细胞与心肌细胞损伤模型共培养，实验分为以下2组：对照组（上层培养淋巴细胞，下层培养经LPS诱导的心肌细胞，共培养48 h）和LPS-exosome组（上层将淋巴细胞和经LPS诱导的心肌细胞来源外泌体混合培养，下层培养经LPS诱导的心肌细胞）。通过流式细胞术检测心肌细胞凋亡。并用人重组IL-38（Re-IL-38）蛋白处理LPS诱导的脓毒症心肌细胞损伤模型，实验分为以下2组：对照组（心肌细胞+LPS）和重组IL-38组（心肌细胞+IL-38预处理+LPS）。通过流式细胞术检测心肌细胞凋亡，Western blotting法检测心肌细胞中凋亡，PI3K/AKT通路相关蛋白表达水平。结果与正常心肌细胞来源外泌体相比，LPS诱导的心肌细胞来源外泌体能够提高淋巴细胞增殖活力（P<0.05），且淋巴细胞中IL-38 mRNA和蛋白表达水平显著升高（P<0.05）。GEO数据库搜索及差异分析发现miR-1275为LPS诱导脓毒症心肌细胞损伤的关键外泌体；双荧光素酶报告基因结果显示，miR-1275 mimics增加WT-IL-38的荧光素酶活性（P<0.05）。LPS诱导的心肌细胞来源外泌体处理的淋巴细胞与LPS诱导的心肌细胞共培养，能抑制心肌细胞凋亡（P<0.05）。Re-IL-38处理后，心肌细胞凋亡率降低（P<0.05），Bax蛋白表达量降低（P<0.05），Bcl-2、p-PI3K和p-AKT蛋白表达量升高（P<0.05）。结论 LPS诱导的心肌细胞外泌体中的miR-1275介导上调淋巴细胞中IL-38的表达，激活PI3K/AKT通路从而抑制LPS诱导的心肌细胞凋亡。

关键词: 外泌体, 心肌细胞, miR-1275, IL-38, 脓毒症

Abstract:

Objective To investigate the effect of cardiomyocytes-derived exosomes on lipopolysaccharide (LPS)-induced cardiomyocyte injury and its mechanism. Methods Exosomes isolated from rat cardiomyocytes with or without LPS treatment were co-cultured with rat lymphocytes. The lymphocytes with or without exosome treatment were co-cultured with LPS-induced rat cardiomyocytes for 48 h. Cardiomyocyte apoptosis was detected using flow cytometry, and the expressions of apoptosis marker proteins and the PI3K/AKT pathway proteins were detected using Western blotting. The effects of human recombinant IL-38 protein on apoptosis and protein expressions in LPS-induced cardiomyocytes were examined. Results Compared with normal cardiomyocyte-derived exosomes, the exosomes from LPS-induced cardiomyocytes significantly enhanced proliferation and increased mRNA and protein expression levels of IL-38 in rat lymphocytes. Bioinformatics analysis suggested that miR-1275 in the exosome played a key role in LPS-induced cardiomyocyte injury, and in dual luciferase reporter gene assay, miR-1275 mimics significantly increased luciferase activity of WT-IL-38. Co-culture with lymphocytes treated with exosomes from LPS-induced cardiomyocytes significantly inhibited apoptosis of LPS-induced cardiomyocytes. Treatment with recombinant IL-38 also effectively lowered apoptosis rate of LPS-induced cardiomyocytes, reduced cellular expression of Bax protein, and increased the protein expression levels of Bcl-2, p-PI3K and p-AKT. Conclusion miR-1275 in exosomes derived from LPS-induced cardiomyocytes mediates IL-38 up-regulation expression in lymphocytes to activate the PI3K/AKT pathway and inhibit LPS-induced cardiomyocyte apoptosis.

Key words: exosomes, cardiomyocyte, miR-1275, IL-38, sepsis

薄海美, 曹新营, 邢平川, 王志军. 外泌体来源的miR-1275通过上调淋巴细胞中IL-38的表达抑制脓毒症心肌细胞凋亡[J]. 南方医科大学学报, 2025, 45(8): 1608-1615.

Haimei BO, Xinying CAO, Pingchuan XING, Zhijun WANG. Exosome-derived miR-1275 mediates IL-38 upregulation in lymphocytes to suppress lipopolysaccharide-induced apoptosis of myocardial cells in vitro[J]. Journal of Southern Medical University, 2025, 45(8): 1608-1615.

图/表 6

图1 qRT-PCR检测外泌体中miRNA的表达

Fig.1 Expression of miRNAs in exosomes detected by qRT-PCR. *P<0.05 vs miR-556 group.

图2 LPS诱导心肌细胞损伤模型

Fig.2 LPS-induced cardiomyocyte injury model. A: Effect of LPS on proliferation of cardiomyocytes. B: Volcano map. C: Heat map. *P<0.05 vs control group.

图3 含miR-1275的外泌体对淋巴细胞增殖及IL-38 mRNA蛋白表达水平的影响

Fig.3 Effect of exosomes containing miR-1275 on lymphocyte proliferation and mRNA and protein expression levels of IL-38. A: Western blotting for detecting expressions of exosome markers CD63 and TSG101. B: CCK-8 assay for assessing the effect of exosomes on lymphocyte proliferation. C: Expression level of IL-38 protein in lymphocytes detected by Western blotting. Group ① was the control group and group ② was the LPS-exosome group. D: Expression level of IL-38 mRNA in lymphocytes detected by qRT-PCR. E: Dual-luciferase reporter gene assay of the regulatory relationship between miR-1275 and IL-38. *P<0.05 vs control group.

图4 流式细胞术检测LPS诱导心肌细胞源性外泌体对心肌细胞凋亡的影响

Fig.4 Flow cytometry for assessing of the effect of LPS-induced cardiomyocyte-derived exosomes on cardiomyocyte apoptosis. *P<0.05 vs control group.

图5 流式细胞术检测IL-38对心肌细胞凋亡的影响

Fig.5 Effect of IL-38 on cardiomyocyte apoptosis detected by flow cytometry. *P<0.05 vs control group.

图6 Western blotting分析IL-38介导的PI3K/AKT通路对外泌体诱导的心肌细胞凋亡的影响。

Fig.6 Western blotting for assessing the effect of IL-38-mediated PI3K/AKT pathway on exosomes-induced cardiomyocyte apoptosis. A: Expression levels of apoptosis-related proteins Bax and Bcl-2. B: Expression levels of proteins related to the PI3K/AKT pathway. ① Control group; ② Re-IL-38 group. *P<0.05 vs control group.

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